Search

The activity of mitochondrial alpha-ketoglutarate dehydrogenase complex (KGDHC) is severely reduced in human pathologies where oxidative stress is traditionally thought to play an important role, such as familial and sporadic forms of Alzheimer's disease and other age-related neurodegenerative diseases. This minireview is focused on substantial data that were accumulated over the last 2 decades to support the concept that KGDHC can be a primary mitochondrial target of oxidative stress and at the same time a key contributor to it by producing reactive oxygen species. This article is part of a Special Issue entitled 'Mitochondrial function and dysfunction in neurodegeneration'.

4-hydroxynonenal (HNE), a product of omega-6 polyunsaturated fatty acid peroxidation, impairs mitochondrial respiration in vitro by adducting the alpha-ketoglutarate dehydrogenase complex (KGDC) and inhibiting its activity. The present study seeks to define whether aging increases HNE adduction to rat heart KGDC, and whether such adduction impacts KGDC activity. We found that hearts from old rats exhibit significantly (por =0.01) higher HNE-modified mitochondrial proteins when compared with those from young rats. Among these proteins, dihydrolipoamide succinyltransferase, the E2k component of KGDC, was most markedly modified (por =0.01) by HNE with age. As opposed to that seen in vitro, no significant change in electrophoretic mobility or impairment in enzyme activity was observed. On the contrary, KGDC activity increased onefold (por =0.01) in old rats, suggesting that the aging myocardium is not affected by HNE adduction or compensates for such damage. Further analysis revealed that heightened KGDC activity was not due to increased protein content or gene expression, but correlates with a lower Km for alpha-ketoglutarate. Thus, contrary to that observed in vitro, the measurement of HNE-KGDC adduct in rat heart is more relevant as a marker of age-related protein oxidation than a factor of mitochondrial dysfunction.

The activity of a key mitochondrial enzyme, the alpha-ketoglutarate dehydrogenase complex (KGDHC), declines in the brains of patients with neurodegenerative diseases such as Alzheimer's disease, as well as in thiamine-deficient (TD) animals. The decreased activity often occurs without a reduction in enzyme protein, which negates the use of immunocytochemistry to study cellular or regional changes in enzyme activity within the brain. To overcome this limitation, an activity staining method using nitroblue tetrazolium was developed. The histochemical activity staining was standardized in cultured cells. The assay was linear with time and was highly specific for KGDHC. The dark-blue reaction product (formazan) formed a pattern that was consistent with mitochondrial localization. Treatment of the cultured cells with both reversible and irreversible inhibitors decreased formazan production, whereas conventional enzyme assays on cell lysates only revealed loss of KGDHC activity with irreversible inhibitors. The activity staining was also linear with time and highly specific for KGDHC activity in mouse brain sections. Staining occurred throughout the brain, and discrete neuronal populations exhibited particularly intense staining. The pattern of staining differed markedly from the distribution of KGDHC protein by immunocytochemistry. Generalized decreases in the intensity of activity staining that occurred in the TD brains compared to controls were comparable with the loss of KGDHC activity by conventional enzyme assay. Thus, the present study introduces a new histochemical method to measure KGDHC activity at the cellular and regional level, which will be useful to determine changes of in situ enzyme activity.

Succinylphosphonate (SP) is a powerful inhibitor or α-ketoglutarate dehydrogenase (KGD). Methylation of the phosphonate reduces its inhibitory effect. The complex of KGD with SP undergoes a kinetically slow transition similar to the process observed during catalysis. α-Ketoglutarate binds to the enzyme—inhibitor complex, preventing its isomerisation.

The burst of product accumulation during the KGD reaction was investigated. It has been shown not to be the obligatory feature of catalysis, but appears when increasing the enzyme saturation by KG. Structural analogues of KG and the SH-group modification suppress the initial burst without preventing catalysis. The results obtained are in favour of the existence of the regulatory site for binding KG and its structural analogues essential for hysteretic properties of KGD.

Reducing 2 SH-groups of KGD by dihydrolipoate leads to cooperativity in substrate binding. Cooperative properties of KGD in the KGD complex are modulated by NADH. Physiological significance of these observations is discussed.

alpha-Ketoglutarate dehydrogenase was inactivated irreversibly and completely during oxidation of alpha-ketoadipic acid. The inactivation was revealed both in the model system with ferricyanide and in the overall reaction catalyzed by the alpha-ketoglutarate dehydrogenase complex. Neither substrate depletion nor product accumulation induced the inactivation. The results obtained were compared with recent data on the enzyme inactivation during oxidation of alpha-ketoglutaric acid. The differences in the inactivation kinetics observed with the two substrates of the enzyme were analyzed. They seem not to reflect the different mechanisms of the inactivation, but, rather, depend on the changes in the rates of the individual stages of the process.

Two classes of mutant lacking α-ketoglutarate dehydrogenase complex activity were detected by biochemical analysis of strains of Escherichia coli requiring succinate for aerobic growth on glucose minimal medium. One class, designated sucA, lacked the α-ketoglutarate decarboxylase component (E1) whereas the other class, sucB, lacked the dihydrolipoyl transsuccinylase component (E2). Studies with mixed cell-free extracts showed that the overall dehydrogenase activity could be reconstituted from several pairs of sucA plus sucB mutants but not from mixtures of mutants of the same class. Transduction analysis with phage P1 indicated close linkage between the two genes and their frequencies of cotransduction with gal were similar. The order of the two genes was also established as sucA (E1)-sucB(E2)...gal by reciprocal three-point crosses with several pairs of mutants.